Hepatitis C virus (HCV) is a major cause of community-acquired viral hepatitis. Infected individuals are at increased risk of developing chronic liver disease and HCV sequelae are now the most common indication for liver transplantation in the US. We previously demonstrated that HCV could be classified into 6 major genotypes and numerous subtypes. Prototype strains of the various genotypes of HCV have been biologically amplified in chimpanzees, packaged and distributed for use as challenge inocula in studies of passive and active immunoprophylaxis, etc. Full-length cDNA clones of HCV [genotypes 1a (strains H77 and HC-TN), 1b (strains HC-J4 and Con1) and 2a (strain HC-J6)] have been constructed and transcribed RNA used to transmit hepatitis C to chimpanzees by in vivo hepatic transfection. Infectivity pools have been prepared from chimpanzees infected with monoclonal HCV (derived by in vivo transfection with RNA transcripts of infectious cDNA); these have been titered for infectivity in other chimpanzees. With the reagents we have developed we are pursuing a collaborative study of the immunopathogenesis of HCV infections in chimpanzees, a surrogate of man. We have demonstrated that resolution versus progression to chronicity is a function of the host, not the virus, since infections with monoclonal viruses have yielded both results. We have shown that humoral immunity appears not to be important in control of the virus, at least in the control of infection or in preventing reinfection. Antibodies directed against the viral envelope proteins develop in animals that become chronically infected, but apparently not in animals with resolved infections. Intrahepatic cellular immune responses are vigorous in chimpanzees with viral control but their strength does not necessarily predict the final outcome of infection. Also, gene expression analysis of acutely infected chimpanzees with different outcomes identified intrahepatic responses associated with HCV viremia, such as interferon alpha, as well as outcome specific responses associated with clearance, such as genes involved in antigene processing and presentation, the adaptive immune response and interferon gamma induction. HCV infection also influenced genes involved in lipid metabolism. Most recently, we have studied the correlation between host response, virus evolution and outcome in chimpanzees infected with a unique HCV strain associated with severe acute liver disease. Analysis of the entire polyprotein sequence of viruses recovered during the first year of follow-up suggested strong positive selection of variant sequences. Thus, strong host cellular immune responses are closely related to the emergence of new virus variants. However, the emergence of such variants does not necessarily lead to viral persistence even when mutations are frequent or in known T-cell epitopes. Since animals infected with the same HCV strain had mild to severe acute liver disease these studies also suggested that the host response is the principal determinant of the severity of acute HCV. We have demonstrated that sterilizing immunity can be achieved by repeated infection of chimpanzees, but that this sterilizing immunity is strain-specific. However, we have also demonstrated that immunity acquired during acute HCV infection does not necessarily prevent persistent infection, even following rechallenge with the homologous monoclonal virus. Preliminary analyses suggest that the persisting viruses represent immune escape variants. When taken together, these studies will provide an in-depth analysis of humoral versus cellular immune responses to HCV infection, and their association with virus evolution. The availability of infectious cDNA clones of HCV has permitted for the first time a mutational analysis of genomic regions. For example, individual portions of the 3' UTR have been deleted from the full-length genotype 1a clone and the resultant deletion mutant clones inoculated into chimpanzees by intrahapatic transfection. Several regions of the UTR have been identified as critical for in vivo replication of HCV. In other studies we have deleted the hypervariable region 1 (HVR1) of the E2 protein of HCV, the region that contains a neutralization epitope. Surprisingly, the deletion mutant virus was viable but attenuated when transfected into chimpanzees. Adaptive mutations that increased the fitness of this deletion mutant were identified. This study also indicated that HVR1 is not essential for the resolution of infection or the progression to chronicity since both outcomes were observed with HCV lacking HVR1. Most recently, we have performed a mutational analysis of the gene that encodes the p7 protein, a protein without a well-defined function. An analogous protein is found in related pestiviruses. By testing deletion mutants and mutants with point mutations we have demonstrated that p7 is critical for the viability of HCV. By testing chimeras between our genotype 1a and 2a clones we have shown also that p7 contains critical genotype specific sequences. In a collaborative study we have extended the in vivo mutagenesis studies to determine the effect of mutations found to permit replication of a subgenomic replicon derived from the HCV strain Con1 in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes increased significantly by a combination of two adaptive mutations in NS3 and a single mutation in NS5A. However, these cell culture-adaptive mutations influenced in vivo infectivity. Following intrahepatic transfection of chimpanzees, the wild-type Con1 genome was infectious and produced viral titers similar to those produced by other infectious HCV clones. Repeated independent transfections with RNA transcripts of a Con1 genome containing the three adaptive mutations failed to achieve active HCV infection. Furthermore, although a chimpanzee transfected with RNA transcripts of a Con1 genome with only the NS5A mutation became infected, viruses recovered at day 7 had a reversion back to the original Con1 sequence. This study demonstrated that mutations that are adaptive for replication of HCV in cell culture might be highly attenuating in vivo. Since most, if not all, replicons studied contain adaptive mutations specific for Huh-7 cells, our study has important implications for the interpretation of the biological relevance of findings in this in vitro system. In other studies, we have constructed chimeric genomes from infectious cDNA clones of HCV and bovine viral diarrhea virus. These genomes can replicate in transfected cells but the resultant viral products cannot assemble into infectious virus in the absence of helper virus. However, the transfected genome expresses large quantities of structural HCV proteins in susceptible cells. We have constructed an infectious cDNA clone of GB virus-B (GBV-B), a monkey virus that is the closest relative to HCV. Experimental infection with GBV-B results in acute viral hepatitis in tamarins. In addition, we have prepared challenge pools of GBV-B and have determined the infectivity titer of these in tamarins. We have shown that GBV-B can infect owl monkeys, but not chimpanzees, suggesting that this virus is not of human origin. We are currently using the GBV-B tamarin system to study characteristics of the virus that it shares with HCV, a virus that must be studied in chimpanzees. We have demonstrated that sterilizing immunity to GBV-B can be achieved by repeated infection of tamarins and that this sterilizing immunity was not due to a high titer of neutralizing antibodies. By testing deletion mutants of the GBV-B clone in tamarins we have shown that each of the predicted encoded GBV-B proteins are critical for the virus. However, viruses with deletions of specific domains within the 3' UTR were found to be viable. Interestingly, infection with one such deletion mutant produced a persistent infection with chronic hepatitis. All previously reported GBV-B infections in tamarins were resolved. Thus infections with this mutant might permit studies of mechanisms for viral persistence in this surrogate model.